**Fast Surface Hydrophilization via Atmospheric Pressure Plasma Polymerization for Biological and Technical Applications**

**Hana Dvoˇráková, Jan Cech \*, Monika Stupavsk ˇ á, Lubomír Prokeš, Jana Jurmanová, Vilma Buršíková, Jozef Ráhel' and Pavel St'ahel**

Department of Physical Electronics, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic; hana.dvorakoval@mail.muni.cz (H.D.); stupavska@mail.muni.cz (M.S.); prokes@chemi.muni.cz (L.P.); janar@physics.muni.cz (J.J.); vilmab@physics.muni.cz (V.B.); rahel@mail.muni.cz (J.R.); pstahel@physics.muni.cz (P.S.)

**\*** Correspondence: cech@physics.muni.cz

Received: 6 August 2019; Accepted: 30 September 2019; Published: 4 October 2019

**Abstract:** Polymeric surfaces can benefit from functional modifications prior to using them for biological and/or technical applications. Surfaces considered for biocompatibility studies can be modified to gain beneficiary hydrophilic properties. For such modifications, the preparation of highly hydrophilic surfaces by means of plasma polymerization can be a good alternative to classical wet chemistry or plasma activation in simple atomic or molecular gasses. Atmospheric pressure plasma polymerization makes possible rapid, simple, and time-stable hydrophilic surface preparation, regardless of the type and properties of the material whose surface is to be modified. In this work, the surface of polypropylene was coated with a thin nanolayer of plasma-polymer which was prepared from a low-concentration mixture of propane-butane in nitrogen using atmospheric pressure plasma. A deposition time of only 1 second was necessary to achieve satisfactory hydrophilic properties. Highly hydrophilic, stable surfaces were obtained when the deposition time was 10 seconds. The thin layers of the prepared plasma-polymer exhibit highly stable wetting properties, they are smooth, homogeneous, flexible, and have good adhesion to the surface of polypropylene substrates. Moreover, they are constituted from essential elements only (C, H, N, O). This makes the presented modified plasma-polymer surfaces interesting for further studies in biological and/or technical applications.

**Keywords:** polymer surface; polymer modification; deposition; plasma polymer; hydrophilization; superhydrophilic layers; atmospheric pressure plasma; polypropylene; surface free energy

#### **1. Introduction**

Since their first adoption, polymers have found broad uses, ranging from technical applications to biological/medical applications [1–3]. Bulk polymer materials have convenient chemical and mechanical properties, and can be easily fabricated and deployed. But the effort towards the enlargement of the application basis of polymer materials poses demand on the modification of their surface properties. This ranges from improving surface wettability, the functionalization of surfaces, or deposition of barrier layers, to the fabrication of biocompatible surfaces [4–12]. In research focused on biological and/or medical applications of polymers, two principal approaches can be identified. The first one is focused on the synthesis of tailored, "bulk" polymers (see, e.g., [13]). The second is focused on the modification of the surface properties of "standard" polymers (see e.g., [12,14–16]).

We can find research on biological applications studying surfaces, polymers, or composites with (highly) hydrophobic as well as hydrophilic properties [17–20]. And, as stated in [21], discussion is ongoing about, e.g., the dependence of bacterial adhesion upon wettability. The positive influence of the hydrophilization of polymer surfaces on biocompatibility was reported, e.g., in studies on the functionalization of polyestersulfon (PES) membranes for dialysis [15], composites of polymethylmethacrylate and hydroxyapatite in dental implants, [17], and the improvement of cell adhesion on the hydrophilic, plasma-oxidized surface of poly(lactide-co-glycolide) (PLGA) [22]. The positive effect of highly hydrophilic surfaces on the antifouling properties, as well as protein resistance, was also reported [5,23–25]. Such antifouling surfaces can be adopted in, e.g., medical [23] or marine applications, such as coatings or sensors [26,27]. In this paper, we would like to introduce a method for the fast and permanent surface hydrophilization of a polymer surface. The method is based on the plasma deposition of plasma-polymeric nanolayers constituted from essential elements only (C, H, N, O).

Common materials like polyethylene (PE), polypropylene (PP), polyvinylchloride (PVC), or polymethylmethacrylate (PMMA) exhibit rather hydrophobic surface properties. The modification of a polymer surface to gain a hydrophilic nature can be done using wet chemistry methods, which could benefit greatly from plasma surface pre-treatment (e.g. immobilizing standard surfactants [28]). The alternative approach for improving wettability can benefit from the lack of wet-chemistry processing. This approach is based on the utilization of gas discharge (plasma) for surface modifications of solid-state surfaces (see, e.g. [29,30]). Direct plasma treatment in atomic gasses (i.e. oxygen, nitrogen, argon, air, etc.) was reported in works [31–33], where the introduction of hydrophilic functional groups to the surface was reported. The influence of plasma etching on the surface topography was also reported [29,34,35]. However, there is a significant drawback to the described techniques. The resulting wetting properties depend strongly on treated material properties and treatment (discharge) conditions [36]. A more significant disadvantage is the so-called aging effect, which is the gradual disappearance of improved wetting properties due to ongoing post-treatment surface reactions during sample storage in the ambient gas [37]. Highly-stable hydrophilic or super-hydrophilic surfaces can be prepared using plasma, e.g., by nano-texturizing the surface accompanied by the delivery of new hydrophilic functional groups. However, long processing times [38,39] in low-pressure plasmas are necessary for this method. Further methods of surface activation are plasma-initiated graft polymerization [40–46] and plasma polymerization [47–59].

Plasma polymerization is the process of creating a highly-branched polymer by plasma-initiated polymerization of the gas precursor. Typically, plasma polymer is created as a thin layer consisting of short chains with random organization and a high degree of crosslinking [48,60,61]. Low pressure plasma deposition systems were first developed for the preparation of homogeneous and uniform organosilicon, halocarbon, or hydrocarbon plasma polymer thin films. The list of investigated applications includes optical, anti-reflection, abrasion-resistant, and low-surface energy coatings, barrier layers, contact lubricants, dielectric layers, or intermediate adhesive and anticorrosive layers [56,62–67]. Low deposition and production rates at low pressure plasmas have motivated the current interest in developing more efficient methods for plasma polymerization using atmospheric pressure (AP) discharges. Depositions at atmospheric pressure allow easy, fast, and continuous processing due to the application of open systems without the need of an expensive vacuum system. The maximum deposition rates of AP systems can be as high as several tens of nm per second [68]. The main drawbacks of AP systems are inadequate uniformity of the deposited films and high precursor consumption.

Even an extremely thin plasma-deposited polymeric layer should be sufficient to provide a significant increase in surface wettability. Therefore, in combination with a suitably-selected AP system, unusually short deposition times can be achieved to significantly improve the aging-free wettability of the coated surface.

The presented fast and cost-effective method for preparing highly hydrophilic surfaces is based on short-time plasma polymerization at atmospheric pressure. We used a low concentration of propane-butane (P-B) diluted in nitrogen as a carrier gas. This gas mixture was known to increase the adhesion of polyester cords to rubber matrices [69]. In addition, this gas mixture is not toxic, nor is it very inexpensive. Deposited thin films are stable, flexible, and they are constituted from the essential elements only (C, H, N, O). Therefore, the presented method can be utilized in biological or technical applications. As potential applications, we can mention the cost-effective production of intermediate layers, biocompatible surfaces that could be utilized in medical applications (e.g. bandages, plasters, device coatings), and marine/water management applications (biofouling control coatings).

The wettability of deposited thin films was characterized by means of the water contact angle (WCA) measurement. The surface free energy (SFE) of thin films was derived from measurements of the WCA and diiodomethane contact angle (DCA). X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy using attenuated total reflection (FTIR-ATR) were used to determine the chemical composition of the deposited layers. Scanning electron microscopy (SEM) imaging was used to characterize the surface morphology.

#### **2. Materials and Methods**

Commercial polypropylene (PP-H, TUPLEX, Brno, Czech Republic) foil with a thickness of 2 mm, manufactured by extrusion, was selected as a model substrate. Polypropylene (PP) samples with surface areas of 25 <sup>×</sup> 95 mm<sup>2</sup> were washed with isopropyl alcohol and dried for 72 hours at room temperature before processing. The water contact angle of the untreated sample was 92◦ and its surface free energy was 31.9 mJ.m<sup>−</sup>2. A commercial mixture of propane and butane (mass percent composition: 84% propane, 15% butane, and 1% of C2 and C5 hydrocarbons) admixed into nitrogen with a purity of 99.999% was used in this study.

Figure 1 depicts the experimental configuration of the plasma deposition setup and sample position during the plasma treatment. The flow and composition of the processing gas were controlled by two mass flow meters. The volume concentration of P-B was set at 0.4, 0.8, and 1.2%, and a total gas flow of 3 l.min−<sup>1</sup> was kept in all cases. The processing gas mixture was injected into the plasma reactor directly in a so-called T-configuration. The so-called Diffuse Coplanar Surface Barrier Discharge (DCSBD) [70] was used to generate plasma for plasma-polymerization. The plasma was generated as a thin layer above the DCSBD surface, using sine-wave high voltage with a frequency of 30 kHz and with a specific power density of 3.75 W.cm<sup>−</sup>2. The PP foil sample was attached to the holder, a moving with a velocity of 15 cm s−<sup>1</sup> along the DCSBD surface to simulate the conditions of continuous processing. The distance between the sample and the DCSBD electrode was set at 0.1 mm; the treatment times ranged from 0.5 s to 20 s.

**Figure 1.** Schematic representation of the experimental set-up.

The wettability of sample surfaces was determined by measuring the static contact angle using the sessile drop method. The contact angles of two standard liquids (deionized water and diiodomethane) were measured and the surface free energy was evaluated by the Owens, Wendt, Rabel, and Kaelble (OWRK) model [71,72]. Sessile drop of a volume of 1 μl was dropped onto the measured surface and analyzed using See System *E* (Advex Instruments, Brno, Czech Republic). The sessile drop projection was acquired and the contact angles (CAs) were determined. Then, a statistical analysis of the contact angle data, including a surface energy evaluation, was performed using the selected OWRK model. Sets of 10–15 drops of each test liquid/surface combination were used for statistical processing and, prior to CA analysis, the sessile drop was allowed to reach CA equilibrium (ca 30 s).

The chemical composition of the untreated and plasma-treated samples was evaluated via FTIR-ATR and XPS. These analyses were performed on samples treated for 15 seconds. Infrared spectra were obtained with FTIR spectrometer Tensor 27 equipped with a single reflection diamond ATR accessory Platinum ATR (Bruker Optics, Ettlingen, Germany). The XPS measurements were performed on the ESCALAB 250Xi (Thermo Fisher Scientific, Waltham, MA, USA). The X-Ray source was a micro-focused monochromatic Al Kα X-Ray source operating at 200 W (650 microns spot size). The measurements were done under the conditions of 50 eV pass energy and a resolution of 1 eV for a survey and of 20 eV pass energy and a resolution of 0.1 eV for high-resolution spectra. The analysis was carried under an ultrahigh vacuum of 10-9 mbar, at room temperature. To avoid surface charging, an electron neutralizer was used. All binding energies were referenced with respect to C-C/C-H at a binding energy of 284.8 eV.

The mechanical properties of the layers were studied by means of nanoindentation using a Hysitron TI950 (Bruker Corporation, Billerica, MA, USA) nanoindenter equipped with a Berkovich diamond indenter. Qualitative bending tests were carried out on coated PP foils to study the bending resistance of the samples. The procedure of the qualitative bending test is shown in Figure 2.

**Figure 2.** Illustration of the qualitative bending test: pictures (**a**) to (**c**) shows the bending procedure.

The surface morphology before and after the bending test was studied using a Tescan MIRA 3 scanning electron microscope. All samples were coated with a thin conductive carbon layer prior to the observation. Images were taken at magnifications of 50,000 and 100,000 times at a working voltage of 15 kV. Study of the sample surface after the bending test allowed us to study the bending resistance as well as the adhesion of the deposited layers to the PP substrate. To measure the film thickness, scalpel sections were made in the most deformed areas of the layers after bending, which enabled a partial removal of the layer from the substrate surface. The thickness was then measured using the SEM on the side portions of the fragments.

#### **3. Results and Discussion**

The presence of a highly-hydrophilic plasma polymer layer was evident already after short processing times. Figure 3 shows the water contact angle (WCA) and surface free energy (SFE) values as a function of the deposition time for three different processing gas compositions (precursor volume concentration 0.4, 0.8 and 1.2%). The value of WCA dropped sharply already during the first 7 seconds of plasma treatment, then remained constant. Surprisingly, WCA values for samples deposited from the lower P-B concentration in nitrogen converged to the final value more rapidly than in the case of higher precursor deposition mixtures. The values of WCA and SFE of samples deposited for 4 seconds or more were almost independent of P-B concentration. Longer deposition times caused only an increase in the layer thickness. For thicker layers, the scatter of the measured values of WCA and SFE decreased, suggesting that the homogeneity of the deposited layer was increased.

**Figure 3.** Dependences of surface parameters values on the plasma treatment time: (**a**) water contact angle (WCA); (**b**) surface free energy (SFE). Values for three different compositions of processing gas, i.e. 0.4, 0.8, and 1.2% of P-B in nitrogen, are shown.

The minimum WCA of 8.7◦ and the maximum SFE of 77 mJ.m−<sup>2</sup> were obtained for 20 seconds of plasma treatment in a processing gas of 1.2% precursor concentration. Nevertheless, a WCA value of about 9◦ was achieved already after 10 seconds of plasma treatment for all tested precursor concentrations. According to [73–75], the criteria for superhydrophilic properties are as follows: either a complete wetting of the surface occurs, or the WCA is close to zero on such surfaces. The latter means, in practice, that the WCA is too low for optical recognition, i.e. according to [73–75], the WCA < 5–10◦. According to these criteria, the prepared surfaces could be classified as superhydrophilic. The SFE components, as well as the diiodomethane contact angles, are given in the supplemental data, Figures S1–S4.

Figure 4 illustrates the visual appearance of the significant decrease of WCA from 92◦ to 9◦ after 15 seconds of plasma treatment in nitrogen with a 0.8% admixture of P-B. Without plasma polymerization, the typical values of WCA for plasma-activated polypropylene were within the range of 20◦ to 70◦ [76–86]. In general, plasma polymerization makes it possible to achieve much lower values of WCA. For example, the plasma polymerization of acrylic acid (AA), one of the most widely used precursors for hydrophilic modifications, produces surfaces with WCA values of 8◦ to 43◦ [45,52–55]. Using a plasma-initiated graft polymerization of AA on polyethylene terephthalate, a surface with a WCA of 5◦ with great time stability was prepared; however, the whole treatment procedure took more than five hours [44]. In this context, our results obtained by plasma deposition from the P-B mixture were very encouraging.

**Figure 4.** The images of water sessile drop on: (**a**) untreated sample; and (**b**) on the sample after 15 seconds of plasma treatment. Images shown for a processing gas with a volume concentration of P-B of 0.8%**.**

Figure 5 shows the aging characteristics of WCA (a) and SFE (b) for samples treated for technologically-feasible times of 4 seconds. The changes were monitored for 21 days of storage under ambient atmosphere conditions. The WCA increased by 2–3◦ and the SFE decreased by 4–6 mJ.m−<sup>2</sup> during the first 7 days of aging; afterwards, their values remained constant. This shows great time stability, especially when compared to the pure plasma activation, in which the wettability improvement decays within a few hours or days [78,79,84,86]. The comparison with other plasma-polymerized or plasma-initiated grafted layers revealed at least equal or slightly better aging characteristics of our P-B layer [40,44,45,59]. The aging of plasma-modified surfaces is a well-known phenomenon described, for example, in [87]. The reversion of the plasma-modified surface properties towards unmodified values was found for both types of modification, i.e., hydrophilization and hydrophobization (see, e.g. [86,88,89]). The aging effect is explained in [87], in which Johansson stated in that thermodynamically driven reorientation, migration of low-molecular-weight additives from the bulk of the plastic to the surface, and airborn contamination of surface should be considered for the aging effect. For the presented plasma polymer thin films, the latter dominates the SFE decrease, explaining the observed decrease from the SFE maximum value being only 7%; see Figure 5. Another mechanism was also reported, i.e., a correlation of surface charges and wetting properties was found [90]. The aging of the surface modification could therefore be influenced also by the plasma-modified electrical properties of the surfaces [91–93].

**Figure 5.** Changes of values (**a**) WCA and (**b**) SFE due to aging effect. Values for three different compositions of processing gas, i.e., 0.4, 0.8, and 1.2% of P-B in nitrogen, are shown. A treatment time of 4 seconds was kept in all cases.

ATR-FTIR analysis was made on plasma-treated and untreated samples. Spectra were normalized between the minimum and maximum intensity values. The normalized spectra of untreated and plasma-treated samples are compared in Figure 6. In the spectra were identified typical C-H absorption bands related to an isotactic polypropylene substrate [94,95]: 2950, 2917, 2837, 1453, 1376, 1359, 1329, 1304, 1167, 1153, 997, 972, 940, 899, 840, and 808 cm<sup>−</sup>1. In the infrared spectra of the treated samples, two additional wide spectral bands were found. The first one, between 3500 and 3000 cm<sup>−</sup>1, is related to hydroxyl groups of carboxylic acids and a N-H group of amines or amides [96,97]. The second band, between 1730 and 1650 cm−1, reflects the presence of carbonyl group of aldehydes, ketones, carboxylic acids and amides, conjugated carbonyls, and imines. Carbonyl groups are often observed in the infrared spectra of polypropylene treated in air plasma [98–100] or in methane/oxygen plasma [101]. They can also originate from the post-treatment effect due to atmospheric humidity [57,95,100,102]. The same functional groups were also identified in the XPS spectra.

**Figure 6.** Infrared spectrum of untreated and plasma-treated samples for varied compositions of processing gas, i.e., 0.4, 0.8, and 1.2% of P-B in nitrogen. A treatment time of 15 seconds was kept in all cases.

Due to a high penetration depth (>1 μm) and low layer thickness (maximum around 70 nm), the increase of nitrogen and oxygen group absorption with the increase of precursor concentration is most likely related more to the increase in layer thickness than to real chemical changes in the plasma polymer (the layer thickness is discussed below, see Table 3).

A considerably better detection of surface chemistry is provided by XPS. This method revealed plasma-induced changes in the PP surface composition with a probing depth of 3–10 nm. The estimated minimum film thickness after 15 seconds of plasma treatment was 15 nm; therefore, we assume that the properties of the plasma polymer were measured without the effect of the underlying PP substrate. Atomic compositions for reference and plasma-modified PP surfaces are presented in Table 1. The XPS survey spectrum of untreated PP surface shows that the dominant signals are from C and O, see Figure S5. The initial PP polymer surface contains a low quantity of oxygen (7 at %).

**Table 1.** Atomic concentrations (at %) and element ratios of untreated and plasma-modified surfaces for three different compositions of processing gas, i.e., 0.4, 0.8, and 1.2% of P-B in nitrogen. XPS spectra were measured immediately after plasma treatment and after 14 days of aging in ambient air. A treatment time of 15 seconds was kept in all cases. The percentages shown are the mean values of three determinations.


The plasma modification resulted in the reduction of the C1s peak to approx. 70%. As the concentration of the precursor increased, the intensity of the C peak decreased, and the nitrogen content increased, indicating the growth of the nitrogen containing surface layer. The XPS data of all PP modified surfaces show nitrogen incorporation into the deposited carbon layer. The most pronounced difference in the surface composition of the untreated PP sample and the coated PP surface is the appearance of a significant N1s peak, see Figure S5.

The high resolution C1s peak was fitted with 6 principal components: C–C/C–H (binding energy at 284.8 eV), C–N (285.7 eV), C–O (286.3 eV), C=N (287.1 eV), C=O (287.8 eV), and O–C=O/C–CO–N (289.1 eV), which is consistent with the FTIR-ATR measurements and previous reports [103,104]. The results of the fits are presented in Table 2 and Figure 7. As shown, the most significant changes after the plasma treatment for PP are the significant increase of the C–N component and the decrease of the C–C/C–H component. The plasma modification resulted in a massive reduction of the hydrocarbon peak to 35% for 1.2% of P-B. Components which correspond to hydrophilic functional groups containing nitrogen (C–N, C=N and C=O–N) were introduced on the PP surface by means of aforementioned plasma modification.

**Table 2.** Percentage (%) of peak areas (functional groups) in C1s peaks of untreated and plasma-modified surfaces for three different compositions of processing gas, i.e., 0.4, 0.8, and 1.2% of P-B in nitrogen. XPS spectra were measured immediately after plasma treatment and after 14 days of aging in ambient air. A treatment time of 15 seconds was kept in all cases.


**Figure 7.** C1s high resolution scan of: (**a**) untreated sample; and (**b**) sample treated for 10 seconds in processing gas with volume concentration of precursor of 0.8%.

The most significant change, i.e., 36%, was seen in the increase in the C–N component (1.2% P-B). In the case of plasma-treated PP samples, the peak areas of C–O, C=O, and O–C=O all increased by a factor 1.5–2 compared to the untreated PP. The relatively low oxygen content is not surprising, since the processing gas did not contain oxygen. Oxygen can be incorporated into the structure of the plasma polymer due to post-treatment reactions with ambient atmosphere or air humidity [68,70], or it may be due to a contamination which occurred while using the open plasma reactor. Table 2 shows that the proportion of C–C/C–H bonds decreased with increasing concentrations of precursor in the processing gas, while the proportion of C–N bonds increased. These results correspond to the WCA (Figure 3a); however, it must be noted that the differences in the value of WCA were very small, i.e., within the measurement errors.

As can be seen from the data in Tables 1–3, the composition and surface chemistry did not change significantly in the two weeks after plasma treatment, which is in a good agreement with the time stability of the wetting properties (see Figure 4).

**Table 3.** Dependence of thickness of coatings of samples deposited for 20 s and calculated deposition rate on the volume concentration of P-B in processing gas.


Figure 8 shows SEM images representing the changes in the surface morphology after plasma treatment and after the bending test. Fine structures on the surface are an artefact from the carbon coating. The surface of plasma-treated samples was smooth, homogenous, and without pinholes or protrusions for all treatment times and for all concentrations of precursor. In fact, there were no differences between the surface topography of the untreated and plasma-treated samples (Figure 8a–e). On the other hand, significant differences were observed between samples subjected to the bending test (Figure 8f–j). The untreated sample showed only a local accumulation of material on the surface and the creation of wrinkles. In contrast, the layers deposited for 20 seconds were intensively wrinkled (and partially cracked) after the severe bending test (see Figure 2). The degree of wrinkling and cracking increased with the concentration of precursor, as shown in Figure 8h–j, mainly because of the increase in film thickness. No cracks were observed on the sample deposited for 7 seconds at 0.4% P-B concentration; however, significant wrinkles were created on the surface after the bending test. With an increase of P-B concentration, the degree of wrinkling and cracking increased, as the film thickness became comparable to 20 s deposition conditions; see Figure S6 and deposition rates in Table 3.

**Figure 8.** Surface topography of untreated and plasma-treated surface before the bending test; top row (**a**) to (**e**); and after the bending test: bottom row (**f**) to (**j**). The treatment time was 7, resp. 20 seconds and the composition of processing gas was 0.4, 0.8, resp. 1.2% of P-B in nitrogen.

The layers were not delaminated from the PP substrate except for the regions scratched with the scalpel (Figure 9a–c). The fragments separated by the scalpel (Figure 9d–f) were used for the measurement of layer thickness and for the linear estimation of the average deposition rate. The measured thickness of coatings, growth rate, and the concentration of precursor are summarized in Table 3.

**Figure 9.** Surface topography of the scratched area for varied composition of processing gas, i.e., 0.4, 0.8, resp. 1.2% of P-B in nitrogen. A treatment time of 20 seconds was kept in all cases. The bottom row represents a magnified view of the surface from the area indicated on the corresponding top row images.

The prepared thin plasma polymer layer is smooth and homogeneous, as shown in Figure 8. The layer thickness increased with the processing time and precursor concentration. The peel test showed that the layer exhibited excellent adhesion to the substrate and, in the case of the lower thickness, is relatively flexible and resistant to delamination and cracking, even in the case of severe mechanical stress.

#### **4. Conclusions**

A new hydrophilization technique based on a plasma deposition of a thin film from mixtures of propane-butane with nitrogen at atmospheric pressure was proposed and successfully tested. Unlike simple plasma treatment, the observed high surface free energy values are due to the properties of the deposited plasma-polymer nanolayer. Therefore, the wettability improvement does not depend on the substrate material, and the aging of the surface modification is highly reduced. The deposited polymer-like layers have proven to be very homogeneous, showing uniform thickness. The measured surface free energy of the coatings was in the range of 60–77 mJ.m<sup>−</sup>2, depending on the coating process conditions. FTIR spectra showed the organic film structure and the presence of C–N and C–C/C–H bonds, as well as C–O, C=O, and O–C=O bonds. The results of XPS analysis were in good accordance with the FTIR observations, confirming also the presence of C–O, C=O, and O–C=O bonds. The basic character of the film surface determined by the surface energy measurement agrees well with the nitrogen-containing hydrophilic groups detected in the surface structure of the films. The films exhibited homogeneous coverage of polymeric substrate and highly hydrophilic properties. With WCA < 10◦, the films could be classified as superhydrophilic [73–75]. Moreover, the hydrophilized surfaces were composed of essential elements only (C, N, O, H), and as such, their properties could be of interest for utilization in biological, as well as technical, applications.

**Supplementary Materials:** The supplementary material is available online at http://www.mdpi.com/2073-4360/ 11/10/1613/s1, Figure S1: Dependences of diiodomethane contact angle (DCA) values on the plasma treatment time. Values given for three different compositions of processing gas, i.e. 0.4, 0.8 and 1.2% of P-B in nitrogen are shown, Figure S2: Dependences of surface free energy (SFE) values and corresponding LW, resp. AB components

*Polymers* **2019**, *11*, 1613

on the plasma treatment time for a gas mixture of 0.4% of P-B in nitrogen, Figure S3: Dependences of surface free energy (SFE) values and corresponding LW, resp. AB components on the plasma treatment time for a gas mixture of 0.8% of P-B in nitrogen, Figure S4: Dependences of surface free energy (SFE) values and corresponding LW, resp. AB components on the plasma treatment time for a gas mixture of 1.2% of P-B in nitrogen, Figure S5: XPS survey spectra of the surface of 'as-received' and after thin film deposition for 10 seconds in a gas mixture of 0.8% of P-B in nitrogen is given. The background of the spectra used for computational peak analysis is provided, Figure S6 Surface topography of plasma-treated sample after bending test is given for 1.2% P-B in nitrogen and 7 seconds deposition time.

**Author Contributions:** In alphabetical order: Conceptualization, V.B., J.R. and P.S.; Formal analysis, J.J., L.P. and M.S.; Funding acquisition, V.B. and P.S.; Investigation, V.B., J.C., H.D., J.J., L.P., M.S. and P.S.; Methodology, H.D., ˇ L.P., J.R. and P.S.; Supervision, P.S.; Writing—original draft, V.B., J.C. and H.D.; Writing—review & editing, V.B., ˇ J.C. and P.S. re. ˇ

**Funding:** This research has been supported by the project LO1411 (NPU I) funded by Ministry of Education Youth and Sports of Czech Republic and by Czech Science Foundation under project GACR 19-15240S.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Electrospinning of Hyaluronan Using Polymer Coelectrospinning and Intermediate Solvent**

**Lenka Vítková 1, Lenka Musilová 1,2, Eva Achbergerová 3, Antonín Minaˇrík 1,2, Petr Smolka 1,2, Erik Wrzecionko 1,2 and Aleš Mráˇcek 1,2,\***


Received: 9 August 2019; Accepted: 8 September 2019; Published: 18 September 2019

**Abstract:** In the current study, we present methods of sodium hyaluronate, also denoted as hyaluronan (HA), nanofiber fabrication using a direct-current (DC) electric field. HA was spun in combination with poly(vinyl alcohol) (PVA) and polyethylene oxide (PEO) and as a pure polymer. Nonaggressive solvents were used due to the possible use of the fibers in life sciences. The influences of polymer concentration, average molecular weight (*M*w), viscosity, and solution surface tension were analyzed. HA and PVA were fluorescent-labeled in order to examine the electrospun structures using fluorescence confocal microscopy. In this study, two intermediate solvent mixtures that facilitate HA electrospinning were found. In the case of polymer co-electrospinning, the effect of the surfactant content on the HA/PVA electrospinning process, and the effect of HA *M*<sup>w</sup> on HA/PEO nanofiber morphology, were examined, respectively.

**Keywords:** electrospinning; hyaluronan; poly(vinyl alcohol); polyethylene oxide; nanofibers; intermediate solvent; fluorescence confocal microscopy

#### **1. Introduction**

Electrospinning, nowadays a well-established fiber-fabrication method first described in 1902 by Cooley and Morton [1,2], is based on several electrohydrodynamic phenomena [3]. The method utilizes Taylor cones, the product of electric field-induced instabilities in liquid bodies stabilized by capillary forces. However, if electric forces overcome capillary forces, a liquid jet is ejected, which is then subjected to elongation at high rates, causing a decrease in diameter up to micron fractions. Due to the large specific surface of the polymer jet, rapid evaporation of the solvent occurs, leading to the solidification of the polymer jet in the form of a nanofiber. Several instabilities may occur and disrupt the electrospinning process, causing particle formation (so-called electrospraying), bead-on-string structure formation, or branching. The preliminary cause of these phenomena is Rayleigh instability, which is surface tension-driven and electrostatically hindered. Similarly to Taylor cone formation prior to electrospinning, cone-shaped undulations may be formed on the cylindrical jet, leading to jet collapse if the charge per unit area is small, providing electrospraying or bead-on-string structured fibers, or undulation stabilization and elongation, giving branched fibers [3,4]. This technique inherently gives nonwoven mats composed of infinite fibers. In practice, DC electric fields of up to tens of kilovolts magnitude are used in most cases.

Nanofibrous materials have uses in many industry fields, for example, in porous materials [5–7], the fuel cell industry [8,9], petroleum engineering [10,11], and particularly biomedical applications such as wound dressing [12,13], drug delivery, [14,15], or tissue engineering [16,17]. The most popular synthetic polymers for electrospinning include polyethylene oxide (PEO), poly(lactide), and polyethyleneimine [4,18,19]. Regarding natural polymers, proteins, such as silk fibroin [20], or polysaccharides, such as alginate, cellulose, or chitosan, can be used [14]. The presence of a nanostructure was proven to enhance cell proliferation [21], which is why combined 3D printing and electrospinning techniques are widely studied for potential uses in tissue engineering (see, e.g., Mori et al. (2018) [22]).

Hyaluronan (HA) is a polysaccharide abundant in the extracellular matrix of living organisms. Its primary structure is linear, and its secondary structure is typically a twisted ribbon. Due to a rather stiff backbone chain caused by the disaccharide structure, internal hydrogen bonds, and interactions with solvents, the ternary structure is an expanded random coil [23,24]. It was experimentally proven that the presence of ions can influence coil diameter [25]. The coil structure is capable of absorbing approximately 1000 times its weight of water [26]. This provides solutions of HA with extraordinarily high viscosity at low concentrations, as well as shear-thinning behavior. Its excellent biocompatibility and solubility in water makes it a popular choice in biomedical applications [27]. HA melt processing is impossible due to its instability at high temperatures [28].

The main complications in HA solution electrospinning are the high surface tension of HA aqueous solutions, extremely high viscosity at low concentrations, preventing the formation of highly concentrated solutions, and the low evaporation rate of water. Attempts to fabricate HA nanofibers using electrospinning have been made by many researchers. A common approach to electrospinning of polymers with low spinnability is the use of a highly spinnable polymer that then serves as a dragging polymer [20,29]. Under certain conditions, this approach provides core–shell nanofibers, as was demonstrated by Ma et al. (2017) [30] using chitosan and an HA solution. To overcome the problem of high surface tension, the use of surfactants [31] or a different solvent choice is possible [27,32,33]. According to Malkin et al. (2017), a change in solvent also has a positive effect on spinnability due to introducing a polymer–solvent demixing solidification mechanism [34]. Previously, it was assumed that electrospinning is only possible above critical concentration, i.e., polymer concentration corresponding to one entanglement per chain [3]. However, using solutions of PEO and polyethylene glycol (PEG), Yu et al. (2006) demonstrated that electrospinning is possible below critical concentration [35]. It was argued that the ability to form smooth fibers via electrospinning is governed by solution elasticity [36–38]. On the other hand, Shenoy et al. (2005) performed electrospinning experiments on several polymer solutions, and concluded that complete stabilization of the electrospinning process is provided by a minimum of 2.5 entanglements per chain [39]. Malkin et al. (2017) argued that stabilization of the electrospinning process can be achieved at concentrations below critical if an intermediate solvent is used [34]. Experiment evidence suggests great contribution of solution elasticity, interaction parameters, surface tension, and conductivity to the electrospinning process. Ambient parameters, such as temperature and humidity, need to be taken into account as well [40]. Although there have been attempts for analysis of electrospinning jet behavior [4,41], so far none are comprehensive enough to account for all influence.

In the current study, nanofibrous mats containing HA were obtained using electrospinning. Two approaches were employed: HA co-electrospinning in a blend with highly spinnable polymers PVA and PEO, respectively, and the use of an intermediate solvent. As intermediate solvents, mixtures containing water and isopropanol (IPA), and water, ethanol EtOH, and methanol (MeOH) were used. The solvent mixtures were found with the aid of a Teas graph, incorporating the method described in Luo et al. (2010) [42]. We attempted to offer insight on the influence of shear viscosity and polymer-chain conformation in the solution on the electrospinning process. Furthermore, HA and PVA were fluorescent-labeled, which allowed the products to be observed by fluorescence confocal microscopy.

#### **2. Materials and Methods**

#### *2.1. Materials and Chemicals*

HA of *M*<sup>w</sup> 243 kDa, 370 kDa, 600 kDa, and 1180 kDa was purchased from Contipro a.s. Demineralized (DEMI) water was prepared using the Milipore Direct-Q 3UV system. PVA of *M*<sup>w</sup> 89–98 kDa, 99+% hydrolyzed, PEO of *M*w 300 and 600 kDa, respectively, EtOH absolute Spectranal, IPA puriss p.a., ACS reagent, disodium hydrogen phosphate dodecahydrate ≥99%, 4-acetamido-TEMPO free radical, 97%, dimethyl sulfoxide (DMSO) ACS reagent, ≥99,9%, Nile Blue A, dye content ≥75%, NaBH3CN reagent grade, 95%, pyridine anhydrous 99.8%, dibutiltin dilaurate, 95%, fluorescein isothyocyanite isomer (FITC), ≥90% and benzethonium chloride (BEC), ≥97% were purchased from Sigma Aldrich. MeOH p.a. was purchased from Lach:Ner. Sodium bromide pure was purchased from Lachema a.s. Sodium hypochlorite solution pure was purchased from Penta. NaCl PharmaGrade was purchased from SAFC. NaHCO3 ACS Grade was purchased from VWR.

#### *2.2. HA Fluorescent Labeling*

In order to prepare Nile Blue A labeled-HA (600 kDa), HA was initially oxidized according to a previously published method, Huerta-Angeles et al. (2012) [43], followed by fluorescent labeling described in Šmejkalová et al. (2017) [44].

Initially, HA (1 g) was dissolved in 10 mL of DEMI water. To the HA solution, sodium bromide (0.129 g) and disodium hydrogen phosphate (0.771 g) were added. The reaction mixture was cooled to 5 ◦C, followed by the addition of 4-acetamido-TEMPO (5 mg) and 450 μL of sodium hypochlorite. The reaction was carried out for 45 min under nitrogen atmosphere at 5 ◦C. The oxidized HA was dialyzed against DEMI water for 3 days and freeze-dried (yield: 96%). In the second step, an aqueous solution (2 wt.%) of oxidized HA (0.5 g) was stirred with Nile Blue A (92 mg) predissolved in DMSO (5 mL) for 5 h. Subsequently, NaBH3CN (79 mg) was added to this reaction mixture which was then stirred over night at room temperature. The crude product was precipitated by NaCl solution and IPA, and the remaining Nile Blue A was washed out using IPA. The product was then dialyzed against 0.5 wt.% NaCl and 0.5 wt.% NaHCO3 aqueous solutions for 2 days and against DEMI water for 3 days. The final product was obtained in a form of a blue lyophilisate (yield 78%).

#### *2.3. PVA Fluorescent Labeling*

PVA was labeled with FITC following the procedure published by Kaneo et al. (2005) [45]. Briefly, PVA (2.5 g) was dissolved in DMSO (66.6 mL) and pyridine (416.6 μL) under stirring at 80 ◦C for 24 h. FITC (83 mg) and dibutiltin dilaurate (31 μL) were added to the PVA solution and the reaction was carried out for 2 h at 95 ◦C in darkness. The crude product was precipitated and washed with IPA, followed by dialysis against DEMI water and lyophilization. The yield of the reaction was 88%.

#### *2.4. Solutions Preparation*

HA of respective *M*<sup>w</sup> was dissolved in binary and ternary solvent mixtures at 50 ◦C under vigorous stirring for 48 h regardless the HA *M*<sup>w</sup> and solvent mixture, to obtain completely homogenized solution. The solvent mixtures chosen for the experiments were H2O:IPA in 10:7 weight ratio, and H2O:EtOH:MeOH in 5:5:1 weight ratio.

HA/PVA blend solutions with BEC surfactant were prepared in the following way; 2 wt.% HA 600 kDa solution and 1 wt.% PVA 89–98 kDa solution were prepared separately by dissolving the respective polymers in DEMI water for 24 h at elevated temperature (50 ◦C for HA, and 80 ◦C for PVA). BEC aqueous solutions of the following concentrations: 1 wt.%, 2 wt.%, 5 wt.%, and 10 wt.% were prepared separately as well. The final solutions were prepared by mixing 2.5 g of HA solution with 2 g of PVA solution. After the components were properly mixed, 0.03 g of BEC solution of respective concentration was added and the solution was mixed properly. Slight turbidity appearance was

observed upon the surfactant addition. For the purpose of confocal microscopy, 4% of the respective polymer content was replaced by fluorescent labeled analogue.

HA/PEO 2 wt.% blend solutions were prepared by mixing the polymers at 1:1 weight ratio and dissolving them in DEMI water by stirring vigorously at room temperature for 48 h. HA *M*w used were 243, 370, and 600 kDa. PEO *M*w used were 300 and 600 kDa. For the purpose of confocal microscopy, 4% of HA content was replaced by Nile Blue A labeled HA.

#### *2.5. Electrospinning Equipment*

A homemade electrospinner consisting of high DC voltage power supply Spellman SL150, a grounded metal collector, 40.3 mm in diameter, and a simple metal rod spinneret, 8 mm in diameter, were used in the study (see Figure 1). The tip-to-collector distance was kept at 76 mm. Experiments were conducted in air atmosphere at room temperature and humidity, and normal pressure. The fibers were collected using a recycled paper substrate to ensure good adhesion.

**Figure 1.** Electrospinning device: (**a**) device scheme and (**b**) device used in experiments.

#### *2.6. Characterization*

Dynamic viscosity was determined using a Malvern Kinexus Pro+ rotational rheometer with cup-and-bob geometry. The measurements were conducted at 25 ◦C at 11 different shear rates ranging from 0.1 to 10 s<sup>−</sup>1.

Portable conductometer Mettler Toledo Seven2Go Pro was used to determine the conductivity of the solutions. Each solution was measured 3 times at room temperature.

Surface tension was determined by a pendant drop method using a Krüss Drop Shape Analyzer DSA 100. Three separate drops of each sample were measured. Each drop was measured 30 times with a 1 s delay between the measurements. Dixon's Q-test was used to exclude the outliers. The measurement was conducted at 25 ◦C in an air atmosphere.

The fiber morphology analysis was done using a Phenom Pro X Scanning Electron Microscope (SEM) in the backscattered electron mode. The samples were sputtered with a layer of gold prior to the analysis. Acceleration voltage was 10 kV. Optical analysis of the images was done using ImageJ software.

An Olympus FLUOVIEW FV3000 Laser Scanning Microscope was used for fluorescence confocal microscopy. Excitation wavelenghts available were 405, 488, 561, and 640 nm. Wavelength ranges 600–640 nm and 450–520 nm were, respectively, used as emission spectra for HA labeled by Nile Blue A, and PVA labeled by FTIC. The immersion objective (*Z* = 60) with numerical aperture *A* = 1.35 was used for nanofibers observation.

#### **3. Results and Discussion**

The electrospinning process is highly dependent on the intrinsic properties of the spinning solution. The most prevalent were polymer *M*w, concentration and polydispersity, all of which were reflected in viscosity, and also surface tension and conductivity [40]. Higher conductivity was presumed to facilitate stability in the spinning process [4], while high surface tension prevented electrospinning onset [3]. Measurement of shear viscosity was done at low shear rates in a narrow range, since the formation of a Taylor cone in sufficiently conductive fluids typically occurs without inducing high shear rates [46], and solutions are considered Newtonian liquids in this part of the process.

#### *3.1. HA/PVA Blend Aqueous Solutions*

In the past, electrospinning HA/PVA aqueous solutions was not possible without the addition of a small amount of surfactant. BEC was chosen due to the coil-shrinking effect on HA conformation [24], which we assumed to be beneficial in terms of electrospinning.

The surface tension of HA/PVA blend aqueous solutions was lower than the HA aqueous solutions (around 70 mN·m−1, see Jurošková (2017) [47]), which is likely the result of surfactant BEC addition. Surface tension decreases with increasing of BEC content. Solution conductivity was increased by the increase of BEC content as a result of BEC ionic nature (see Table 1).

**Table 1.** Characteristics of Hyaluronan (HA)/Poly(vinyl alcohol) (PVA) blend solution with benzethonium chloride (BEC).


HA/PVA blend aqueous solutions show the highest conductivity of the spinnable solutions used in the current study (see Tables 1 and 3–5). This is likely the synergic effect of dissociation of HA and PVA in water, and the addition of ionic surfactant to the solution.

Low polymer concentration causes the viscosity of the solutions to be low as well. As apparent from Figure 2, there was a quick drop of viscosity present upon addition of 0.065 wt.% of BEC.

**Figure 2.** Viscosity of HA/PVA blend solutions with BEC as a function of shear rate.

0.065 wt.% of BEC was close to critical aggregation concentration [24] and HA coil shrinkage was expected, causing significant increase in turbidity of the solution, which was observed during the preparation, and it was in agreement with findings of Gˇrundˇelová et al. (2013) [24]. It is safe to assume that BEC effectively created an intermediate solvent to HA, and electrospinning HA is therefore encouraged not only by mixing with highly spinnable PVA, but also by the intermediate solvent effect. Due to HA precipitation, further increase of BEC content would be counterproductive.

The effect of surfactant content on electrospun-structure morphology and the electrospinning process was examined. As the BEC content increased, the minimum spinning voltage decreased (see Table 2) due to the decrease in surface tension.


**Table 2.** Morphology analysis of HA/PVA blend solutions with BEC electrospinning products.

With the increase of BEC content, product morphology shifted from elongated beads to bead-on-string structured fibers (see Figure 3).

**Figure 3.** Scanning Electron Microscopy (SEM) micrographs of electrospun structures obtained from HA/PVA blend solutions with BEC. BEC content (**a**) 0.033 wt.%. and (**b**) 0.065 wt.%.

The increase in stability may be the result of increased conductivity of the solution, as argued by Reneker and Yarin (2008) [4], or the increase in polymer–polymer interactions, i.e., lowering solvent quality, which would be in agreement with the findings of Malkin et al. (2017) [34]. However, electrospinning smooth fibers was not achieved by this method. There were multiple reasons, such as insufficient solution elasticity, difference in HA and PVA viscoelastic behavior, or uneven BEC distribution, leading to formation of clumps of the respective polymers. Higher BEC content also led to the formation of multiple Taylor cones, therefore increasing the yield of the process. To proceed on this subject, it would be possible to choose a different surfactant with higher critical aggregation concentration, or alter the HA/PVA ratio in the solution in a way to increase the elongation elasticity. Both approaches would certainly lead to a better understanding of the co-electrospinning phenomenon, and might lead to smooth nanofiber production. The electrospun structures did not exceed 1 μm in diameter (see Table 2).

Nile Blue A labeled HA 600 kDa and FITC labeled PVA 89–98 kDa blend aqueous solution containing 0.065 wt.% of BEC was electrospun, and the products were observed by fluorescence confocal microscopy. It is clear from Figure 4 that both polymers are present jointly in fibers, as well as beads. However, due to insufficient magnification it was not possible to determine the respective position of the polymers within the structures. The absence of some structures when illuminated by a different wavelength suggested fluctuation in the contents of the respective polymers throughout spinning.

(**b**)

**Figure 4.** *Cont.*

(**c**)

**Figure 4.** Fluorescence confocal microscope micrographs of electrospun structures obtained from Nile Blue A labeled HA 600 kDa and FITC labeled PVA 89–98 kDa aqueous solution with BEC content 0.065 wt.%. (**a**) Nile Blue A labeled HA visible. Emission spectrum 600–640 nm. (**b**) FITC labeled PVA visible. Emission spectrum 450–520 nm. (**c**) Both fluorescent labeled polymers visible—combined emission spectra.

#### *3.2. HA/PEO Blend Aqueous Solutions*

HA/PEO blend solutions were spun in order to produce HA containing nanofibers from aqueous solutions without use of any additional substances, such as surfactants or salts. PEO served as elasticity and shear viscosity mediator, as well as a highly spinnable polymer for co-electrospinning. In order to examine the influence of HA *M*<sup>w</sup> on the electrospinning process, PEO was used in such *M*<sup>w</sup> and concentration that would facilitate the electrospinning of all HA *M*<sup>w</sup> chosen. The PEO *M*<sup>w</sup> and concentration were found experimentally.

Surface tension of HA/PEO blend aqueous solutions was significantly higher than the one of HA in intermediate solvents solutions (see Tables 3–5) due to use of water as a solvent, but still considerably lower than HA aqueous solutions [47], caused by the surface tension-enhancing effect of HA being hindered by the presence of PEO, which induced a decrease in surface tension of the aqueous solutions instead [48].


**Table 3.** Characteristics of HA/Polyethylene oxide (PEO) aqueous blend solutions.

The effect of respective polymers *M*<sup>w</sup> on the surface tension was inconclusive, as a result of the low polymer concentration used (2 wt.%). Conductivity of the solutions was significantly higher than that of the pure HA solutions (see Tables 3–5), despite the lower concentration of HA. This can be attributed to the higher dissociation of HA in water than in solvents containing alcohols. As a result of the increase in molar fraction with the decrease in *M*<sup>w</sup> while the same weight fraction of a polymer was kept, solution conductivity slightly decreased with the increase of HA *M*w. As the *M*w decreased, the effect of the end groups also gained significance and, in the case of HA and PEO, contributed to conductivity as well.


**Table 4.** Characteristics of HA solutions in H2O:Isopropanol (IPA) in 10:7 weight ratio solvent mixtures.

**Table 5.** Characteristics of HA solutions in H2O:Ethanol (EtOH):Methanol (MeOH) in 5:5:1 weight ratio solvent mixtures.


Solution viscosity dropped as the *M*<sup>w</sup> of the respective polymers decreased (see Figure 5). Viscosity was significantly lower than that in the case of solutions of HA in intermediate solvents, which was one of the purposes of adding PEO into an HA aqueous solution.

**Figure 5.** Viscosity of HA/PEO blend solutions as a function of shear rate.

In order to examine the effect of HA *M*<sup>w</sup> on the morphology of structures electrospun from HA/PEO blend aqueous solutions, the processing parameters, i.e., tip-to-collector distance and spinning voltage, were kept constant for each series of samples. All of the solutions gave bead-on-string structured fibers, which was a result of Raileygh instability acting on a conductive liquid jet in a strong electric field [4]. The structures did not exceed 1 μm in size (see Table 6).



As can be seen in Figure 6, the shape of the beads was influenced by the *M*<sup>w</sup> of PEO. If PEO 300 kDa (Figure 6a–c) was used, the beads were almost spherical, while for PEO 600 kDa (Figure 6d–f) strong elongation of the beads was apparent.

(**c**) (**d**) **Figure 6.** *Cont.*

**Figure 6.** SEM micrographs of electrospun structures obtained from HA/PEO blend solutions. (**a**) HA 243 kDa, PEO 300 kDa. (**b**) HA 370 kDa, PEO 300 kDa. (**c**) HA 600 kDa, PEO 300 kDa. (**d**) HA 243 kDa, PEO 600 kDa. (**e**) HA 370 kDa, PEO 600 kDa. (**f**) HA 600 kDa, PEO 600 kDa.

The significance of morphology difference could even suggest different bead origin. Another explanation is a significant shift in solution elasticity induced by the different *M*<sup>w</sup> of PEO. A great difference in viscoelasticity of the respective polymers also contributed to the uneven distribution of polymers throughout the spinning via a phenomenon known as polymer wrapping in coextrusion [49]. Further experimental examination of this phenomenon is needed in order to fully understand the causes. No effect of HA *M*w on HA/PEO fiber morphology was found in the current study, as it was likely hindered by the significant PEO content in the used samples.

Electrospinning of HA/PEO blend solution containing Nile Blue A labeled HA 600 kDa and PEO 600 kDa allowed us to investigate the obtained structures by using fluorescence confocal microscopy. This technique proved the presence of HA in both fibers and beads (see Figure 7). On account of PEO's nonfluorescence, it is not visible in the figure.

**Figure 7.** Fluorescence confocal microscope micrographs of electrospun structures obtained from the Nile Blue A Labeled HA 600 kDa and PEO 600 kDa aqueous solution. Emission spectrum: 600–640 nm.

#### *3.3. HA Solutions in Intermediate Solvents*

Two solvent mixture systems were chosen for the experiments: H2O:IPA in 10:7 weight ratio and H2O:EtOH:MeOH in 5:5:1 weight ratio. These were chosen with the aid of a Teas graph (see Figure 8) in such manner that they would lower the surface tension compared to water solutions, and encourage polymer–polymer interactions over polymer–solvent interactions, leading to smooth nanofiber production.

Electrospinning solutions of three different concentrations were considered for each solvent mixture and HA *M*w, respectively, with the intention to find upper and lower limiting concentration for electrospinning.

**Figure 8.** Solvent–mixture representation in Teas graph. 1: water; 2: IPA; 3: MeOH; 4: EtOH; 5: H2O:IPA 10:7; 6: H2O:EtOH:MeOH 5:5:1.

The use of H2O:IPA an H2O:EtOH:MeOH mixed solvents led to a significant reduction of surface tension compared to the aqueous HA solutions, see Tables 4 and 5 [47]. H2O:EtOH:MeOH solutions showed slightly higher surface tension than that of the H2O:IPA solutions.

No significant effect of HA concentration or *M*<sup>w</sup> on surface tension was found in the current study, which was due to very low polymer concentrations and the narrow range of concentrations used. As a consequence of HA's ionic nature, solution conductivity was decreased as the concentration of the polymer was decreased. Electrospinning at very low concentration could therefore be hindered by two mechanisms—insufficient polymer chain entanglement, caused by low polymer concentration, and instability of the cylindrical jet, due to a decrease in conductivity.

The shear viscosity of the solutions decreased with the decrease in both concentration, and *M*w of the polymer, with the difference being in the range of several orders of magnitude, as is evident from Figure 9.

**Figure 9.** Viscosity of HA solutions in intermediate solvents as a function of shear rate. (**a**) H2O:IPA in 10:7 weight ratio solvent mixture. (**b**) H2O:EtOH:MeOH in 5:5:1 weight ratio solvent mixture.

Viscosity is sometimes considered the determining parameter of spinnability via electrostatic force [18]. The findings of this study contradict such assumption as overly simplified, which is in agreement with Yarin et al. (2001) [46], who claimed that shear viscosity was insignificant in terms of Taylor cone formation. The maximum viscosity of a spinnable solution can differ as much as ten times if different *M*w of the same polymer are used. We assumed that the determining parameter was polymer chain entanglement, which is affected by the polymer chain conformation in given solvent, the ionic strength of the solution, polymer concentration, and other parameters. Further investigation on this subject is necessary.

Regardless the solvent mixture, spinning voltage decreased with the decrease of concentration (see Tables 7 and 8), which can possibly be explained by a shift in solution viscoelasticity, causing the critical instability wavelength leading to Taylor cone formation to increase [3], thus consuming less energy and lowering spinning voltage. Since surface tension does not change with concentration, its effect on spinning voltage can be neglected.

**Table 7.** Morphology analysis of H2O:IPA in 10:7 weight ratio HA solutions electrospinning products.


**Table 8.** Morphology analysis of H2O:EtOH:MeOH in 5:5:1 weight ratio HA solutions electrospinning products.


No influence of the concentration on the electrospun structure morphology was found, which was the result of little difference in the concentrations of the respective samples. However, the concentration clearly governed the transition between electrospinning and electrospraying, which is explained by polymer chain entanglement according to Shenoy et al. (2005) [39] or by polymer solution elasticity according to Yu et al. (2006) [35]. The mechanism could not clearly be determined from the experiments conducted in the current study. The obtained fibers did not exceed 100 nm in diameter in any of the cases, making them promising in terms of biomedicine. The spherical particle diameter was mostly in the 1 to 0.1 μm range.

In the case of each solvent mixture, HA 600 kDa showed more tendency to undergo instabilities, which resulted in a combination of electrospinning and electrospraying (Figures 10a–c and 11a–b), whereas HA 1180 kDa was able to provide smooth fibers (Figures 10d and 11c,d). It could be assumed that a higher HA *M*w was more favorable in terms of electrospinning as a result of the higher elasticity of the solution.

**Figure 10.** SEM micrographs of electrospun structures obtained from H2O:IPA in 10:7 weight ratio solutions. (**a**) 3.2 wt.% HA *M*w 600 kDa; (**b**) 2.3 wt.% HA *M*w 600 kDa; (**c**) 1.3 wt.% HA *M*w 600 kDa; (**d**) 2.9 wt.% HA *M*w 1180 kDa; (**e**) 1.2 wt.% HA *M*w 1180 kDa.

**Figure 11.** SEM micrographs of electrospun structures obtained from H2O:EtOH:MeOH in 5:5:1 weight ratio solutions. (**a**) 2.8 wt.% HA *M*w 600 kDa; (**b**) 2.4 wt.% HA *M*w 600 kDa; (**c**) 2.2 wt.% HA *M*w 1180 kDa; (**d**) 1.5 wt.% HA *M*w 1180 kDa.

We can also see that instabilities in the form of electrospraying and branching were more frequent for solutions using H2O:IPA in 10:7 weight ratio solvent mixture (Figure 10). Solvent mixture H2O:EtOH:MeOH in 5:5:1 weight ratio provided higher stability to the electrospinning process. As the conductivity of the solutions using respective solvent mixtures was comparable (see Tables 4 and 5), the stabilization mechanism was presumably more complex than stabilization by charge density suggested by Reneker and Yarin (2008) [4]. Polymer chain conformation and interaction parameters can be expected to have a significant influence on electrospinning phenomena stabilization, but further investigation is necessary in order to fully understand the process.

Although the conductivity of HA solutions in intermediate solvents was significantly lower than those of aqueous blend solutions with PVA or PEO, smooth fibers were only obtained from a certain HA in intermediate solvent solutions. Our conclusion is that although conductivity does have a positive effect on electrospun jet stabilization, as stated by Reneker and Yarin (2008) [4], the influence of polymer viscoelasticity needs to be taken into account, as suggested by Stepanyan et al. (2014) and Palangetic et al. (2014) [36,37]. In the case of blend-solution electrospinning, the situation is complicated due to the difference in the viscoelastic properties of the polymers, which are simultaneously drawn at high elongation rates. A non-negligible effect of interaction parameters was present as well, because an intermediate solvent has a great influence on the solution behavior in a strong electric field, as was demonstrated in this study, as well as previously [34,39,42].

#### **4. Conclusions**

Electrospinning of biocompatible and biodegradable polymers is a desirable technique for use in biomedicine and life sciences. Production of HA nanofibers is a challenging task due to the extremely high viscosity and high surface tension of aqueous solutions.

In this study, nanofibers containing HA were obtained by solution electrospinning. Two approaches to the problem were chosen: co-electrospinning of aqueous blend solutions of HA/PVA and HA/PEO, respectively, and use of the intermediate solvent for pure HA solutions electrospinning. The choice of materials was done with regard to potential uses for cell cultivation. To facilitate fiber formation in HA/PVA blend solutions, the addition of BEC was necessary. Both HA/PEO and HA/PVA blend solutions provided bead-on-string structured fibers. As intermediate solvents, H2O:IPA in a 10:7 weight ratio and H2O:EtOH:MeOH in a 5:5:1 weight ratio were chosen. Both solvent mixtures facilitate the electrospinning of HA of *M*<sup>w</sup> 600 and 1180 kDa. Lower *M*<sup>w</sup> solutions had higher tendency to form spherical particles. There is clear correlation between the decrease in solution surface tension and the decrease in spinning voltage can be seen in the results, but no significant impact of these parameters on the fiber diameter was found. Variation in electrospun-structure dimensions and morphology was intensely associated with the change in *M*<sup>w</sup> of the polymers. It was experimentally demonstrated that shear viscosity cannot be used as a sole determining parameter of solution spinnability, as there are differences as high as ten times the order of magnitude for spinnable solutions that differ only in polymer *M*w.

The best results were achieved with the HA 1180 kDa solution in H2O:EtOH:MeOH 5:5:1 at concentrations of 2.2 wt.% and 1.5 wt.%, as these provided smooth fibers. Fiber diameter did not exceed 100 nm for any sample that provided fibers,which makes them promising in terms of tissue engineering.

**Author Contributions:** Conceptualization, L.V. and A.M. (Aleš Mráˇcek); methodology, L.V., L.M., E.A., P.S., E.W., and A.M. (Antonín Minaˇrík); validation, L.V. and A.M. (Aleš Mráˇcek); formal analysis, L.V.; investigation, L.V. and A.M. (Aleš Mráˇcek); resources, L.V. and A.M. (Aleš Mráˇcek); writing—original draft preparation, L.V.; writing—review and editing, L.V. and A.M. (Aleš Mráˇcek); visualization, L.V. and A.M. (Aleš Mráˇcek); supervision, A.M. (Aleš Mráˇcek)

**Funding:** The research was funded by the Ministry of Education, Youth, and Sports of the Czech Republic, Program NPU I (LO1504); the European Regional Development Fund (No. CZ.1.05/2.1.00/19.0409); as well as by TBU (Nos. IGA/FT/2018/011 and IGA/FT/2019/012), funded from resources for specific university research. The work of author Eva Achbergerová was supported by the European Regional Development Fund under project CEBIA-Tech Instrumentation No. CZ.1.05/2.1.00/19.0376.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **Abbreviations**

The following abbreviations are used in this manuscript:


#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*
